Long lasting synthetic glucagon like peptide {GLP-!}

ABSTRACT

Modified insulinotropic peptides are disclosed. The modified insulinotropic peptides are capable of forming a peptidase stabilized insulinotropic peptide. The modified insulinotropic peptides are capable of forming covalent bonds with one or more blood components to form a conjugate. The conjugates may be formed in vivo or ex vivo. The modified peptides are administered to treat humans with diabetes and other related diseases.

This application claims the benefit under 35 U.S.C. §119(e) of UnitedStates provisional patent application No. 60/159,783 filed Oct. 15,1999, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to modified insulinotropic peptides. Inparticular, this invention relates to modified glucagon like peptidesand exendin peptides with long duration of action for the treatment ofdiabetes and other insulinotropic peptide related diseases,gastrointestinal function and activities associated with glucagonlevels.

BACKGROUND OF THE INVENTION

The insulinotropic peptide hormone glucagon-like peptide (GLP-1) hasbeen implicated gas a possible therapeutic agent for the management oftype 2 non-insulin-dependent diabetes mellitus as well as relatedmetabolic disorders, such as obesity. Other useful insulinotropicpeptides include exendin 3 and exendin 4. While useful, GLP-1, exendin 3and exendin 4 suffer from limited duration of action associated withshort plasma half-lifes in vivo, mainly due to rapid serum clearance andproteolytic degradation. The enzyme responsible for the degradation ofGLP-1, dipeptidyl peptidase IV, has been identified. Extensive work hasbeen done in attempts to inhibit the peptidase or to modify GLP-1 insuch a way that its degradation is slowed down while still maintainingbiological activity. Despite these extensive efforts, a long lasting,active GLP-1 has not been produced. As such, the diabetic community hasa tremendous need for improved GLP-1, exendin 3 and exendin 4 peptides.

There is thus a need to modify GLP-1, exendin 3, exendin 4 and otherinsulinotropic peptides to provide longer duration of action in vivo,while maintaining their low toxicity and therapeutic advantages.

SUMMARY OF THE INVENTION

In order to meet those needs, the present invention is directed tomodified insulinotropic peptides (ITPs). This invention relates to novelchemically reactive derivatives of insulinotropic peptides that canreact with available functionalities on cellular carriers includingmobile blood proteins to form covalent linkages. Specifically, theinvention relates to novel chemically reactive derivatives ofinsulinotropic peptides such as glucagon like peptide (GLP) and exendin3 and exendin 4 that can react with available functionalities on mobileblood proteins to form covalent linkages. The invention also relates tonovel chemically reactive derivatives or analogs of insulinotropicpeptides that can react with available functionalities on mobile bloodproteins to form covalent linkages.

The present invention relates to modified insulinotropic peptidescomprising a reactive group which reacts with amino groups, hydroxylgroups or thiol groups on blood compounds to form stable covalent bonds.

The present invention relates to an insulinotropic hormone comprising amodified fragment of GLP-1 and derivatives thereof, especially GLP-1(7-36) amide. The invention additionally pertains to the therapeuticuses of such compounds, and especially to the use of modified GLP-1(7-36) amide for the treatment of maturity onset diabetes mellitus (typeII diabetes).

The present invention further relates to modified Exendin 3 and Exendin4 fragments and therapeutic uses of such compounds.

In particular, the present invention is directed to GLP-1 (1-36)-Lys³⁷(ε-MPA)-NH₂; GLP-1 (1-36)-Lys³⁷ (ε-AAEA-AEEA-MPA)-NH₂; GLP-1(7-36)-Lys³⁷ (ε-MPA)-NH₂; GLP-1 (7-36)-Lys³⁷⁻(ε-AEEA-AEEA-MPA)-NH₂;D-Ala⁸ GLP1 (7-36)-Lys³⁷ (ε-MPA)-NH₂; Exendin-4 (1-39)-Lys⁴⁰(ε-MPA)-NH₂; Exendin-4 (1-39)-Lys⁴⁰ (ε-AEEA-AEEA-MPA)-NH₂; Exendin-3(1-39)-Lys⁴⁰ (ε-MPA)-NH₂; Exendin-3 (1-39)-Lys⁴⁰ (ε-AEEA-AEEA-MPA)-NH₂;Lys²⁶(ε-MPA)GLP-1 (7-36)NH₂; GLP-1 (7-36)-EDA-MPA and Exendin-4(1-39)-EDA-MPA.

The present invention further relates to compositions comprising thederivatives of the insulinotropic peptides and the use of thecompositions for treating diabetes in humans.

The invention further pertains to a method for enhancing the expressionof insulin which comprises providing to a mammalian pancreatic Beta-typeislet cell an effective amount of the modified insulinotropic peptidesdisclosed above.

The invention further pertains to a method for treating maturity-onsetdiabetes mellitus which comprises administration of an effective amountof the insulinotropic peptides discussed above to a patient in need ofsuch treatment.

The invention further pertains to the treatment of other insulinotropicpeptide related diseases and conditions with the modified insulinotropicpeptides of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

To ensure a complete understanding of the invention the followingdefinitions are provided:

Insulinotropic Peptides: Insulinotropic peptides (ITPs) are peptideswith insulinotropic activity. Insulinotropic peptides stimulate, orcause the stimulation of, the synthesis or expression of the hormoneinsulin. Such peptides include precursors, analogues, fragments ofpeptides such as Glucagon-like peptide, exendin 3 and exendin 4 andother peptides with insulinotropic activity.

Glucagon-Like Peptide: Glucagon-Like Peptide (GLP) and GLP derivativesare intestinal hormones which generally simulate insulin secretionduring hyperglycemia, suppresses glucagon secretion, stimulates (pro)insulin biosynthesis and decelerates gastric emptying and acidsecretion. Some GLPs and GLP derivatives promote glucose uptake by cellsbut do not simulate insulin expression as disclosed in U.S. Pat. No.5,574,008 which is hereby incorporated by reference.

Exendin 3 and Exendin 4 Peptides: Exendin 3 and exendin 4 peptides andpeptide derivatives are 39 amino acid peptides which are approximately53% homologons to GLP-1 and have insulinotropic activity.

Reactive Groups: Reactive groups are chemical groups capable of forminga covalent bond. Such reactive agents are coupled or bonded to aninsulinotropic peptide of interest to form a modified insulinotropicpeptide. Reactive groups will generally be stable in an aqueousenvironment and will usually be carboxy, phosphoryl, or convenient acylgroup, either as an ester or a mixed anhydride, or an imidate, therebycapable of forming a covalent bond with functionalities such as an aminogroup, a hydroxy or a thiol at the target site on mobile bloodcomponents. For the most part, the esters will involve phenoliccompounds, or be thiol esters, alkyl esters, phosphate esters, or thelike. Reactive groups include succinimidyl and maleimido groups.

Functionalities: Functionalities are groups on blood components to whichreactive groups on modified insulinotropic peptides react to formcovalent bonds. Functionalities include hydroxyl groups for bonding toester reactive entities; thiol groups for bonding to malemides andmaleimido groups, imidates and thioester groups; amino groups forbonding to carboxy, phosphoryl or acyl groups on reactive entities andcarboxyl groups for bonding to amino groups. Such blood componentsinclude blood proteins.

Linking Groups: Linking groups are chemical moieties that link orconnect reactive groups to ITPs. Linking groups may comprise one or morealkyl groups such as methyl, ethyl, propyl, butyl, etc. groups, alkoxygroups, alkenyl groups, alkynyl groups or amino group substituted byalkyl groups, cycloalkyl groups, polycyclic groups, aryl groups,polyaryl groups, substituted aryl groups, heterocyclic groups, andsubstituted heterocyclic groups. Linking groups may also comprise polyethoxy aminoacids such as AEA ((2-amino)ethoxy acetic acid) or apreferred linking group AEEA ([2-(2-amino)ethoxy)]ethoxy acetic acid).

Blood Components: Blood components may be either fixed or mobile. Fixedblood components are non-mobile blood components and include tissues,membrane receptors, interstitial proteins, fibrin proteins, collagens,platelets, endothelial cells, epithelial cells and their associatedmembrane and membraneous receptors, somatic body cells, skeletal andsmooth muscle cells, neuronal components, osteocytes and osteoclasts andall body tissues especially those associated with the circulatory andlymphatic systems. Mobile blood components are blood components that donot have a fixed situs for any extended period of time, generally notexceeding 5, more usually one minute. These blood components are notmembrane-associated and are present in the blood for extended periods oftime and are present in a minimum concentration of at least 0.1 μg/ml.Mobile blood components include serum albumin, transferrin, ferritin andimmunoglobulins such as IgM and IgG. The half-life of mobile bloodcomponents is at least about 12 hours.

Protective Groups: Protective groups are chemical moieties utilized toprotect peptide derivatives from reacting with themselves. Variousprotective groups are disclosed herein and in U.S. Pat. No. 5,493,007which is hereby incorporated by reference. Such protective groupsinclude acetyl, fluorenylmethyloxycarbonyl (FMOC), t-butyloxycarbonyl(BOC), benzyloxycarbonyl (CBZ), and the like. The specific protectedamino acids are depicted in Table 1.

TABLE 1 NATURAL AMINO ACIDS AND THEIR ABBREVIATIONS 3-Letter 1-LetterProtected Amino Name Abbreviation Abbreviation Acids Alanine Ala AFmoc-Ala-OH Arginine Arg R Fmoc-Arg(Pbf)-OH Asparagine Asn NFmoc-Asn(Trt)-OH Aspartic acid Asp D Asp(tBu)-OH Cysteine Cys CFmoc-Cys(Trt) Glutamic acid Glu E Fmoc-Glu(tBu)-OH Glutamine Gln QFmoc-Gln(Trt)-OH Glycine Gly G Fmoc-Gly-OH Histidine His HFmoc-His(Trt)-OH Isoleucine Ile I Fmoc-Ile-OH Leucine Leu L Fmoc-Leu-OHLysine Lys K Fmoc-Lys(Mtt)-OH Methionine Met M Fmoc-Met-OH PhenylalaninePhe F Fmoc-Phe-OH Proline Pro P Fmoc-Pro-OH Serine Ser SFmoc-Ser(tBu)-OH Threonine Thr T Fmoc-Thr(tBu)-OH Tryptophan Trp WFmoc-Trp(Boc)-OH Tyrosine Tyr Y Boc-Tyr(tBu)-OH Valine Val V Fmoc-Val-OH

Sensitive Functional Groups—A sensitive functional group is a group ofatoms that represents a potential reaction site on an ITP peptide. Ifpresent, a sensitive functional group may be chosen as the attachmentpoint for the linker-reactive group modification. Sensitive functionalgroups include but are not limited to carboxyl, amino, thiol, andhydroxyl groups.

Modified Peptides—A modified ITP is a peptide that has been modified byattaching a reactive group, and is capable of forming a peptidasestabilized peptide through conjugation to blood components. The reactivegroup may be attached to the therapeutic peptide either via a linkinggroup, or optionally without using a linking group. It is alsocontemplated that one or more additional amino acids may be added to thetherapeutic peptide to facilitage the attachment of the reactive group.Modified peptides may be administered in vivo such that conjugation withblood components occurs in vivo, or they may be first conjugated toblood components in vitro and the resulting peptidase stabalized peptide(as defined below) administered in vivo. The terms “modified therapeuticpeptide” and “modified peptide” may be used interchangeably in thisapplication.

Peptidase Stabilized ITP—A peptidase stabilized ITP is a modifiedpeptide that has been conjugated to a blood component via a covalentbond formed between the reactive group of the modified peptide and thefunctionalities of the blood component, with or without a linking group.Peptidase stabilized peptides are more stable in the presence ofpeptidases in vivo than a non-stabilized peptide. A peptidase stabilizedtherapeutic peptide generally has an increased half life of at least10-50% as compared to a non-stabalized peptide of identical sequence.Peptidase stability is determined by comparing the half life of theunmodified ITP in serum or blood to the half life of a modifiedcounterpart therapeutic peptide in serum or blood. Half life isdetermined by sampling the serum or blood after administration of themodified and non-modified peptides and determining the activity of thepeptide. In addition to determining the activity, the length of the ITPmay also be measured by HPLC and Mass Spectrometry.

DETAILED DESCRIPTION OF THE INVENTION

Taking into account these definitions the focus of this invention is tomodify insulinotropic peptides to improve bio-availability, extendhalf-life and distribution through selective conjugation onto a proteincarrier but without modifying their remarkable therapeutic properties.The carrier of choice (but not limited to) for this invention would bealbumin conjugated through its free thiol by a insulinotropic peptidederivatized with a maleimide moiety.

1. Insulinotropic Peptides

A. GLP-1 and Its Derivatives The hormone glucagon is known to besynthesized as a high molecular weight precursor molecule which issubsequently proteolytically cleaved into three peptides: glucagon,glucagon-like peptide 1 (GLP-1), and glucagon-like peptide 2 (GLP-2).GLP-1 has 37 amino acids in its unprocessed form as shown in SEQ IDNO: 1. Unprocessed GLP-1 is essentially unable to mediate the inductionof insulin biosynthesis. The unprocessed GLP-1 peptide is, however,naturally converted to a 31-amino acid long peptide (7-37 peptide)having amino acids 7-37 of GLP-1 (“GLP-1 (7-37)”) SEQ ID NO:2. GLP-1(7-37) can also undergo additional processing by proteolytic removal ofthe C-terminal glycine to produce GLP-1 (7-36) which also existspredominantly with the C-terminal residue, arginine, in amidated form asarginineamide, GLP-1 (7-36) amide. This processing occurs in theintestine and to a much lesser extent in the pancreas, and results in apolypeptide with the insulinotropic activity of GLP-1 (7-37).

A compound is said to have an “insulinotropic activity” if it is able tostimulate, or cause the stimulation of, the synthesis or expression ofthe hormone insulin. The hormonal activity of GLP-1 (7-37) and GLP-1(7-36) appear to be specific for the pancreatic beta cells where itappears to induce the biosynthesis of insulin. The glucagon-like-peptidehormone of the invention is useful in the study of the pathogenesis ofmaturity onset diabetes mellitus, a condition characterized byhyperglycemia in which the dynamics of insulin secretion are abnormal.Moreover, the glucagon-like peptide is useful in the therapy andtreatment of this disease, and in the therapy and treatment ofhyperglycemia.

Peptide moieties (fragments) chosen from the determined amino acidsequence of human GLP-1 constitute the starting point in the developmentcomprising the present invention. The interchangeable terms “peptidefragment” and “peptide moiety” are meant to include both synthetic andnaturally occurring amino acid sequences derivable from a naturallyoccurring amino acid sequence.

The amino acid sequence for GLP-1 has been reported by severalresearchers (Lopez, L. C., et al., Proc. Natl. Acad. Sci., USA80:5485-5489 (1983); Bell, G. I., et al., Nature 302:716-718 (1983);Heinrich, G., et al., Endocrinol. 115:2176-2181 (1984)). The structureof the preproglucagon mRNA and its corresponding amino acid sequence iswell known. The proteolytic processing of the precursor gene product,proglucagon, into glucagon and the two insulinotropic peptides has beencharacterized. As used herein, the notation of GLP-1 (1-37) refers to aGLP-1 polypeptide having all amino acids from 1 (N-terminus) through 37(C-terminus). Similarly, GLP-1 (7-37) refers to a GLP-1 polypeptidehaving all amino acids from 7 (N-terminus) through 37 (C-terminus).Similarly, GLP-1 (7-36) refers to a GLP-1 polypeptide having all aminoacids from number 7 (N-terminus) through number 36 (C-terminus).

In one embodiment, GLP-1 (7-36) and its peptide fragments aresynthesized by conventional means as detailed below, such as by thewell-known solid-phase peptide synthesis described by Merrifield, J. M.(Chem. Soc. 85:2149 (1962)), and Stewart and Young (Solid Phase PeptideSynthesis (Freeman, San Francisco, 1969), pages 27-66), which areincorporated by reference herein. However, it is also possible to obtainfragments of the proglucagon polypeptide, or of GLP-1, by fragmentingthe naturally occurring amino acid sequence, using, for example, aproteolytic enzyme. Further, it is possible to obtain the desiredfragments of the proglucagon peptide or of GLP-1 through the use ofrecombinant DNA technology, as disclosed by Maniatis, T., et al.,Molecular Biology: A Laboratory Manual, Cold Spring Harbor, N.Y. (1982),which is hereby incorporated by reference.

The present invention includes peptides which are derivable from GLP-1such as GLP-1 (1-37) and GLP-1 (7-36). A peptide is said to be“derivable from a naturally occurring amino acid sequence” if it can beobtained by fragmenting a naturally occurring sequence, or if it can besynthesized based upon a knowledge of the sequence of the naturallyoccurring amino acid sequence or of the genetic material (DNA or RNA)which encodes this sequence.

Included within the scope of the present invention are those moleculeswhich are said to be “derivatives” of GLP-1 such as GLP-1 (1-37) andespecially GLP-1 (7-36). Such a “derivative” has the followingcharacteristics: (1) it shares substantial homology with GLP-1 or asimilarly sized fragment of GLP-1; (2) it is capable of functioning asan insulinotropic hormone and (3) using at least one of the assaysprovided herein, the derivative has either (i) an insulinotropicactivity which exceeds the insulinotropic activity of either GLP-1, or,more preferably, (ii) an insulinotropic activity which can be detectedeven when the derivative is present at a concentration of 10⁻¹⁰ M, or,most preferably, (iii) an insulinotropic activity which can be detectedeven when the derivative is present at a concentration of 10⁻¹¹ M.

A derivative of GLP-1 is said to share “substantial homology” with GLP-1if the amino acid sequences of the derivative is at least 80%, and morepreferably at least 90%, and most preferably at least 95%, the same asthat of GLP-1 (1-37).

The derivatives of the present invention include GLP-1 fragments which,in addition to containing a sequence that is substantially homologous tothat of a naturally occurring GLP-1 peptide may contain one or moreadditional amino acids at their amino and/or their carboxy termini.Thus, the invention pertains to polypeptide fragments of GLP-1 that maycontain one or more amino acids that may not be present in a naturallyoccurring GLP-1 sequence provided that such polypeptides have aninsulinotropic activity which exceeds that of GLP-1. The additionalamino acids may be D-amino acids or L-amino acids or combinationsthereof.

The invention also includes GLP-1 fragments which, although containing asequence that is substantially homologous to that of a naturallyoccurring GLP-1 peptide may lack one or more additional amino acids attheir amino and/or their carboxy termini that are naturally found on aGLP-1 peptide. Thus, the invention pertains to polypeptide fragments ofGLP-1 that may lack one or more amino acids that are normally present ina naturally occurring GLP-1 sequence provided that such polypeptideshave an insulinotropic activity which exceeds that of GLP-1.

The invention also encompasses the obvious or trivial variants of theabove-described fragments which have inconsequential amino acidsubstitutions (and thus have amino acid sequences which differ from thatof the natural sequence) provided that such variants have aninsulinotropic activity which is substantially identical to that of theabove-described GLP-1 derivatives. Examples of obvious or trivialsubstitutions include the substitution of one basic residue for another(i.e. Arg for Lys), the substitution of one hydrophobic residue foranother (i.e. Leu for Ile), or the substitution of one aromatic residuefor another (i.e. Phe for Tyr), etc.

In addition to those GLP-1 derivatives with insulinotropic activity,GLP-1 derivatives which stimulate glucose uptate by cells but do notstimulate insulin expression or secretion are within the scope of thisinvention. Such GLP-1 derivatives are described in U.S. Pat. No.5,574,008.

GLP-1 derivatives which stimulate glucose uptake by cells but do notstimulate insulin expression or secretion which find use in theinvention include:

R₁-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Xaa-Gly-Arg-R₂(SEQ ID NO:3) wherein R₁ is selected from a) H₂N; b) H₂N-Ser; c)H₂N-Val-Ser; d) H₂N-Asp-Val-Ser; e) H₂N-Ser-Asp-Val-Ser (SEQ ID NO:4);f) H₂N-Thr-Ser-Asp-Val-Ser (SEQ ID NO:5); g) H₂N-Phe-Thr-Ser-Asp-Val-Ser(SEQ ID NO:6); h) H₂N-Thr-Phe-Thr-Ser-Asp-Val-Ser (SEQ ID NO:7); i)H₂N-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser (SEQ ID NO:8); j)H₂N-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser (SEQ ID NO:9); or, k)H₂N-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser (SEQ ID NO:10). In thepeptide, X is selected from Lys or Arg and R₂ is selected from NH₂, OH,Gly-NH₂, or Gly-OH. These peptides are C-terminal GLP-1 fragments whichdo not have insulinotropic activity but which are nonetheless useful fortreating diabetes and hyperglycemic conditions as described in U.S. Pat.No. 5,574,008.

B. Exendin 3 and Exendin 4 Peptides

Exendin 3 and Exendin 4 are 39 amino acid peptides (differing atresidues 2 and 3) which are approximately 53% homologous to GLP-1 andfind use as insulinotropic agents.

The Exendin-3 [SEQ ID No:11] sequence is HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS and

The Exendin-4 [SEQ ID No:12] sequence is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS.

The invention also encompasses the insulinotropic fragments of exendin-4comprising the amino acid sequences: Exendin-4 (1-31) [SEQ ID No:13]HGEGTFTSDLSKQMEEAVR LFIEWLKNGGPY and Exendin-4 (1-31) [SEQ ID No:14]HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGY.

The invention also encompasses the inhibitory fragment of exendin-4comprising the amino acid sequence:

Exendin-4(9-39) [SEQ ID No:15] DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

Other insulinotropic peptides as presented in the Examples are shown asSEQ ID NO:16-22.

The present invention includes peptides which are derivable from thenaturally occurring exendin 3 and exendin 4 peptides. A peptide is saidto be “derivable from a naturally occurring amino acid sequence” if itcan be obtained by fragmenting a naturally occurring sequence, or if itcan be synthesized based upon a knowledge of the sequence of thenaturally occurring amino acid sequence or of the genetic material (DNAor RNA) which encodes this sequence.

Included within the scope of the present invention are those moleculeswhich are said to be “derivatives” of exendin 3 and exendin 4. Such a“derivative” has the following characteristics: (1) it sharessubstantial homology with exendin 3 or exendin 4 or a similarly sizedfragment of exendin 3 or exendin 4; (2) it is capable of functioning asan insulinotropic hormone and (3) using at least one of the assaysprovided herein, the derivative has either (i) an insulinotropicactivity which exceeds the insulinotropic activity of either exendin 3or exendin 4, or, more preferably, (ii) an insulinotropic activity whichcan be detected even when the derivative is present at a concentrationof 10⁻¹⁰ M, or, most preferably, (iii) an insulinotropic activity whichcan be detected even when the derivative is present at a concentrationof 10⁻¹¹ M.

A derivative of exendin 3 and exendin 4 is said to share “substantialhomology” with exendin 3 and exendin 4 if the amino acid sequences ofthe derivative is at least 80%, and more preferably at least 90%, andmost preferably at least 95%, the same as that of either exendin 3 or 4or a fragment of exendin 3 or 4 having the same number of amino acidresidues as the derivative.

The derivatives of the present invention include exendin 3 or exendin 4fragments which, in addition to containing a sequence that issubstantially homologous to that of a naturally occurring exendin 3 orexendin 4 peptide may contain one or more additional amino acids attheir amino and/or their carboxy termini. Thus, the invention pertainsto polypeptide fragments of exendin 3 or exendin 4 that may contain oneor more amino acids that may not be present in a naturally occurringexendin 3 or exendin 4 sequences provided that such polypeptides have aninsulinotropic activity which exceeds that of exendin 3 or exendin 4.

Similarly, the invention includes exendin 3 or exendin 4 fragmentswhich, although containing a sequence that is substantially homologousto that of a naturally occurring exendin 3 or exendin 4 peptide may lackone or more additional amino acids at their amino and/or their carboxytermini that are naturally found on a exendin 3 or exendin 4 peptide.Thus, the invention pertains to polypeptide fragments of exendin 3 orexendin 4 that may lack one or more amino acids that are normallypresent in a naturally occurring exendin 3 or exendin 4 sequenceprovided that such polypeptides have an insulinotropic activity whichexceeds that of exendin 3 or exendin 4.

The invention also encompasses the obvious or trivial variants of theabove-described fragments which have inconsequential amino acidsubstitutions (and thus have amino acid sequences which differ from thatof the natural sequence) provided that such variants have aninsulinotropic activity which is substantially identical to that of theabove-described exendin 3 or exendin 4 derivatives. Examples of obviousor trivial substitutions include the substitution of one basic residuefor another (i.e. Arg for Lys), the substitution of one hydrophobicresidue for another (i.e. Leu for Ile), or the substitution of onearomatic residue for another (i.e. Phe for Tyr), etc.

2. Modified Insulinotropic Peptides

This invention relates to modified insulinotropic peptides and theirderivatives. The modified insulinotropic peptides of the inventioninclude reactive groups which can react with available reactivefunctionalities on blood components to form covalent bonds. Theinvention also relates to such modifications, such combinations withblood components and methods for their use. These methods includeextending the effective therapeutic in vivo half life of the modifiedinsulinotropic peptides.

To form covalent bonds with the functional group on a protein, one mayuse as a chemically reactive group (reactive entity) a wide variety ofactive carboxyl groups, particularly esters, where the hydroxyl moietyis physiologically acceptable at the levels required to modify theinsulinotropic peptides. While a number of different hydroxyl groups maybe employed in these linking agents, the most convenient would beN-hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS),maleimide-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxysuccinimide ester (GMBS) and maleimidopropionic acid (MPA).

Primary amines are the principal targets for NHS esters as diagramed inthe schematic below.” Accessible α-amine groups present on the N-terminiof proteins react with NHS esters. However, α-amino groups on a proteinmay not be desirable or available for the NHS coupling. While five aminoacids have nitrogen in their side chains, only the ε-amine of lysinereacts significantly with NHS esters. An amide bond is formed when theNHS ester conjugation reaction reacts with primary amines releasingN-hydroxysuccinimide as demonstrated in the schematic below. Thesesuccinimide containing reactive groups are herein referred to assuccinimidyl groups.

In the preferred embodiments of this invention, the functional group onthe protein will be a thiol group and the chemically reactive group willbe a maleimido-containing group such as (GMBA or MPA). GMBA stands forgamma-maleimide-butrylamide. Such maleimide containing groups arereferred to herein as maleido groups.

The maleimido group is most selective for sulfhydryl groups on peptideswhen the pH of the reaction mixture is kept between 6.5 and 7.4. At pH7.0, the rate of reaction of maleimido groups with sulfhydryls is1000-fold faster than with amines. A stable thioether linkage betweenthe maleimido group and the sulfhydryl is formed which cannot be cleavedunder physiological conditions.

The insulinotropic peptides and peptide derivatives of the invention maybe modified for specific labeling and non-specific labeling of bloodcomponents.

A. Specific Labeling

Preferably, the modified insulinotropic peptides (ITP) of this inventionare designed to specifically react with thiol groups on mobile bloodproteins. Such reaction is preferably established by covalent bonding ofa therapeutic peptide modified with a maleimide link (e.g. prepared fromGMBS, MPA or other maleimides) to a thiol group on a mobile bloodprotein such as serum albumin or IgG.

Under certain circumstances, specific labeling with maleimides offersseveral advantages over non-specific labeling of mobile proteins withgroups such as NHS and sulfo-NHS. Thiol groups are less abundant in vivothan amino groups. Therefore, the maleimide derivatives of thisinvention will covalently bond to fewer proteins. For example, inalbumin (the most abundant blood protein) there is only a single thiolgroup. Thus, ITP-maleimide-albumin conjugates will tend to compriseapproximately a 1:1 molar ratio of IP to albumin. In addition toalbumin, IgG molecules (class II) also have free thiols. Since IgGmolecules and serum albumin make up the majority of the soluble proteinin blood they also make up the majority of the free thiol groups inblood that are available to covalently bond to maleimide-modified ITPs.

Further, even among free thiol-containing blood proteins, specificlabeling with maleimides leads to the preferential formation ofITP-maleimide-albumin conjugates, due to the unique characteristics ofalbumin itself. The single free thiol group of albumin, highly conservedamong species, is located at amino acid residue 34 (Cys³⁴). It has beendemonstrated recently that the Cys³⁴ of albumin has increased reactivityrelative to free thiols on other free thiol-containing proteins. This isdue in part to the very low pK value of 5.5 for the Cys³⁴ of albumin.This is much lower than typical pK values for cysteines residues ingeneral, which are typically about 8. Due to this low pK, under normalphysiological conditions Cys³⁴ of albumin is predominantly in theionized form, which dramatically increases its reactivity, as reportedin. In addition to the low pK value of Cys³⁴, another factor whichenhances the reactivity of Cys³⁴ is its location, which is in a creviceclose to the surface of one loop of region V of albumin. This locationmakes Cys³⁴ very available to ligands of all kinds, and is an importantfactor in Cys³⁴'s biological role as free radical trap and free thiolscavenger. These properties make Cys³⁴ highly reactive withITP-maleimides, and the reaction rate acceleration can be as much as1000-fold relative to rates of reaction of TP-maleimides with otherfree-thiol containing proteins.

Another advantage of ITP-maleimide-albumin conjugates is thereproducibility associated with the 1:1 loading of peptide to albuminspecifically at Cys³⁴. Other techniques, such as glutaraldehyde, DCC,EDC and other chemical activations of, for example, free amines lackthis selectivity. For example, albumin contains 52 lysine residues,25-30 of which are located on the surface of albumin and accessible forconjugation. Activating these lysine residues, or alternativelymodifying peptides to couple through these lysine residues, results in aheterogenous population of conjugates. Even if 1:1 molar ratios ofpeptide to albumin are employed, the yield will consist of multipleconjugation products, some containing 0, 1, 2 or more peptides peralbumin, and each having peptides randomly coupled at any one of the25-30 available lysine sites. Given the numerous combinations possible,characterization of the exact composition and nature of each batchbecomes difficult, and batch-to-batch reproducibility is all butimpossible, making such conjugates less desirable as a therapeutic.Additionally, while it would seem that conjugation through lysineresidues of albumin would at least have the advantage of delivering moretherapeutic agent per albumin molecule, studies have shown that a 1:1ratio of therapeutic agent to albumin is preferred. In an article byStehle, et al., “The Loading Rate Determines Tumor Targeting Propertiesof Methotrexate-Albumin Conjugates in Rats,” Anti-Cancer Drugs, Vol. 8,pp. 677-685 (1997), incorporated herein in its entirety, the authorsreport that a 1:1 ratio of the anti-cancer methotrexate to albuminconjugated via glutaraldehyde gave the most promising results. Theseconjugates were taken up by tumor cells, whereas conjugates bearing 5:1to 20:1 methotrexate molecules had altered HPLC profiles and werequickly taken up by the liver in vivo. It is postulated that at thesehigher ratios, conformational changes to albumin diminish itseffectiveness as a therapeutic carrier.

Through controlled administration of maleimide-ITPs in vivo, one cancontrol the specific labeling of albumin and IgG in vivo. In typicaladministrations, 80-90% of the administered maleimide-ITPs will labelalbumin and less than 5% will label IgG. Trace labeling of free thiolssuch as glutathione will also occur. Such specific labeling is preferredfor in vivo use as it permits an accurate calculation of the estimatedhalf-life of the administered agent.

In addition to providing controlled specific in vivo labeling,maleimide-TPs can provide specific labeling of serum albumin and IgG exvivo. Such ex vivo labeling involves the addition of maleimide-ITPs toblood, serum or saline solution containing serum albumin and/or IgG.Once modified ex vivo with maleimide-TPs, the blood, serum or salinesolution can be readministered to the blood for in vivo treatment.

In contrast to NHS-peptides, maleimide-ITPs are generally quite stablein the presence of aqueous solutions and in the presence of free amines.Since maleimide-ITPs will only react with free thiols, protective groupsare generally not necessary to prevent the maleimide-ITPs from reactingwith itself. In addition, the increased stability of the peptide permitsthe use of further purification steps such as HPLC to prepare highlypurified products suitable for in vivo use. Lastly, the increasedchemical stability provides a product with a longer shelf life.

B. Non-Specific Labeling

The ITPs of the invention may also be modified for non-specific labelingof blood components. Bonds to amino groups will generally be employed,particularly with the formation of amide bonds for non-specificlabeling. To form such bonds, one may use as a chemically reactive groupcoupled to the ITP a wide variety of active carboxyl groups,particularly esters, where the hydroxyl moiety is physiologicallyacceptable at the levels required. While a number of different hydroxylgroups may be employed in these linking agents, the most convenientwould be N-hydroxysuccinimide (NHS) and N-hydroxy-sulfosuccinimide(sulfo-NHS).

Other linking agents which may be utilized are described in U.S. Pat.5,612,034, which is hereby incorporated herein.

The various sites with which the chemically reactive groups of thenon-specific ITPs may react in vivo include cells, particularly redblood cells (erythrocytes) and platelets, and proteins, such asimmunoglobulins, including IgG and IgM, serum albumin, ferritin, steroidbinding proteins, transferrin, thyroxin binding protein,α-2-macroglobulin, and the like. Those receptors with which thederivatized ITPs react, which are not long-lived, will generally beeliminated from the human host within about three days. The proteinsindicated above (including the proteins of the cells) will remain in thebloodstream at least three days, and may remain five days or more(usually not exceeding 60 days, more usually not exceeding 30 days)particularly as to the half life, based on the concentration in theblood.

For the most part, reaction will be with mobile components in the blood,particularly blood proteins and cells, more particularly blood proteinsand erythrocytes. By “mobile” is intended that the component does nothave a fixed situs for any extended period of time, generally notexceeding 5 minutes, more usually one minute, although some of the bloodcomponents may be relatively stationary for extended periods of time.Initially, there will be a relatively heterogeneous population oflabeled proteins and cells. However, for the most part, the populationwithin a few days after administration will vary substantially from theinitial population, depending upon the half-life of the labeled proteinsin the blood stream. Therefore, usually within about three days or more,IgG will become the predominant labeled protein in the blood stream.

Usually, by day 5 post-administration, IgG, serum albumin anderythrocytes will be at least about 60 mole %, usually at least about 75mole %, of the conjugated components in blood, with IgG, IgM (to asubstantially lesser extent) and serum albumin being at least about 50mole %, usually at least about 75 mole %, more usually at least about 80mole %, of the non-cellular conjugated components.

The desired conjugates of non-specific ITPs to blood components may beprepared in vivo by administration of the ITPs directly to the patient,which may be a human or other mammal. The administration may be done inthe form of a bolus or introduced slowly over time by infusion usingmetered flow or the like.

If desired, the subject conjugates may also be prepared ex vivo bycombining blood with derivatized ITPs of the present invention, allowingcovalent bonding of the modified ITPs to reactive functionalities onblood components and then returning or administering the conjugatedblood to the host. Moreover, the above may also be accomplished by firstpurifying an individual blood component or limited number of components,such as red blood cells, immunoglobulins, serum albumin, or the like,and combining the component or components ex vivo with the chemicallyreactive ITPs. The labeled blood or blood component may then be returnedto the host to provide in vivo the subject therapeutically effectiveconjugates. The blood also may be treated to prevent coagulation duringhandling ex vivo.

3. Synthesis of Modified ITPs

A. ITP Synthesis

ITP fragments may be synthesized by standard methods of solid phasepeptide chemistry known to those of ordinary skill in the art. Forexample, ITP fragments may be synthesized by solid phase chemistrytechniques following the procedures described by Steward and Young(Steward, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2ndEd., Pierce Chemical Company, Rockford, Ill., (1984) using an AppliedBiosystem synthesizer. Similarly, multiple fragments may be synthesizedthen linked together to form larger fragments. These synthetic peptidefragments can also be made with amino acid substitutions at specificlocations.

For solid phase peptide synthesis, a summary of the many techniques maybe found in J. M. Stewart and J. D. Young, Solid Phase PeptideSynthesis, W.H. Freeman Co. (San Francisco), 1963 and J. Meienhofer,Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (NewYork), 1973. For classical solution synthesis see G. Schroder and K.Lupke, The Peptides, Vol. 1, Acacemic Press (New York). In general,these methods comprise the sequential addition of one or more aminoacids or suitably protected amino acids to a growing peptide chain.Normally, either the amino or carboxyl group of the first amino acid isprotected by a suitable protecting group. The protected or derivatizedamino acid is then either attached to an inert solid support or utilizedin solution by adding the next amino acid in the sequence having thecomplimentary (amino or carboxyl) group suitably protected and underconditions suitable for forming the amide linkage. The protecting groupis then removed from this newly added amino acid residue and the nextamino acid (suitably protected) is added, and so forth.

After all the desired amino acids have been linked in the propersequence, any remaining protecting groups (and any solid support) areremoved sequentially or concurrently to afford the final polypeptide. Bysimple modification of this general procedure, it is possible to addmore than one amino acid at a time to a growing chain, for example, bycoupling (under conditions which do not racemize chiral centers) aprotected tripeptide with a properly protected dipeptide to form, afterdeprotection, a pentapeptide.

A particularly preferred method of preparing compounds of the presentinvention involves solid phase peptide synthesis wherein the amino acidα-N-terminal is protected by an acid or base sensitive group. Suchprotecting groups should have the properties of being stable to theconditions of peptide linkage formation while being readily removablewithout destruction of the growing peptide chain or racemization of anyof the chiral centers contained therein. Suitable protecting groups are9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl,t-amyloxycarbonyl, isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like. The9-fluorenyl-methyloxycarbonyl (Fmoc) protecting group is particularlypreferred for the synthesis of ITP fragments. Other preferred side chainprotecting groups are, for side chain amino groups like lysine andarginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro,p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, andadamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxycarbonyl,2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyland acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; forhistidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl;for tryptophan, formyl; for asparticacid and glutamic acid, benzyl andt-butyl and for cysteine, triphenylmethyl (trityl).

In the solid phase peptide synthesis method, the α-C-terminal amino acidis attached to a suitable solid support or resin. Suitable solidsupports useful for the above synthesis are those materials which areinert to the reagents and reaction conditions of the stepwisecondensation-deprotection reactions, as well as being insoluble in themedia used. The preferred solid support for synthesis of α-C-terminalcarboxy peptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-1%divinylbenzene). The preferred solid support for α-C-terminal amidepeptides is the4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resinavailable from Applied Biosystems (Foster City, Calif.). Theα-C-terminal amino acid is coupled to the resin by means ofN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC)or O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium-hexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT),benzotriazol-1-yloxy-tris(dimethylamino)phosphonium-hexafluorophosphate(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), mediatedcoupling for from about 1 to about 24 hours at a temperature of between100 and 50° C. in a solvent such as dichloromethane or DMF.

When the solid support is4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin,the Fmoc group is cleaved with a secondary amine, preferably piperidine,prior to coupling with the α-C-terminal amino acid as described above.The preferred method for coupling to the deprotected4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resinis O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluoro-phosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. Thecoupling of successive protected amino acids can be carried out in anautomatic polypeptide synthesizer as is well known in the art. In apreferred embodiment, the α-N-terminal amino acids of the growingpeptide chain are protected with Fmoc. The removal of the Fmocprotecting group from the α-N-terminal side of the growing peptide isaccomplished by treatment with a secondary amine, preferably piperidine.Each protected amino acid is then introduced in about 3-fold molarexcess, and the coupling is preferably carried out in DMF. The couplingagent is normallyO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.).

At the end of the solid phase synthesis, the polypeptide is removed fromthe resin and deprotected, either in successively or in a singleoperation. Removal of the polypeptide and deprotection can beaccomplished in a single operation by treating the resin-boundpolypeptide with a cleavage reagent comprising thianisole, water,ethanedithiol and trifluoroacetic acid. In cases wherein theα-C-terminal of the polypeptide is an alkylamide, the resin is cleavedby aminolysis with an alkylamine. Alternatively, the peptide may beremoved by transesterification, e.g. with methanol, followed byaminolysis or by direct transamidation. The protected peptide may bepurified at this point or taken to the next step directly. The removalof the side chain protecting groups is accomplished using the cleavagecocktail described above. The fully deprotected peptide is purified by asequence of chromatographic steps employing any or all of the followingtypes: ion exchange on a weakly basic resin (acetate form); hydrophobicadsorption chromatography on underivitized polystyrene-divinylbenzene(for example, Amberlite XAD); silica gel adsorption chromatography; ionexchange chromatography on carboxymethylcellulose; partitionchromatography, e.g. on Sephadex G-25, LH-20 or countercurrentdistribution; high performance liquid chromatography (HPLC), especiallyreverse-phase HPLC on octyl- or octadecylsilyl-silica bonded phasecolumn packing.

Molecular weights of these ITPs are determined using Fast AtomBombardment (FAB) Mass Spectroscopy.

The ITPs of the invention may be synthesized with N- and C-terminalprotecting groups for use as pro-drugs.

1. N-Terminal Protective Groups

As discussed above, the term “N-protecting group” refers to those groupsintended to protect the α-N-terminal of an amino acid or peptide or tootherwise protect the amino group of an amino acid or peptide againstundesirable reactions during synthetic procedures. Commonly usedN-protecting groups are disclosed in Greene, “Protective Groups InOrganic Synthesis,” (John Wiley & Sons, New York (1981)), which ishereby incorporated by reference. Additionally, protecting groups can beused as pro-drugs which are readily cleaved in vivo, for example, byenzymatic hydrolysis, to release the biologically active parent.α-N-protecting groups comprise loweralkanoyl groups such as formyl,acetyl (“Ac”), propionyl, pivaloyl, t-butylacetyl and the like; otheracyl groups include 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, -chlorobutyryl,benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like;sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-ethoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike; arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl,9-fluorenylmethyloxycarbonyl (Fmoc) and the like and silyl groups suchas trimethylsilyl and the like.

2. Carboxy Protective Groups

As discussed above, the term “carboxy protecting group” refers to acarboxylic acid protecting ester or amide group employed to block orprotect the carboxylic acid functionality while the reactions involvingother functional sites of the compound are performed. Carboxy protectinggroups are disclosed in Greene, “Protective Groups in Organic Synthesis”pp. 152-186 (1981), which is hereby incorporated by reference.Additionally, a carboxy protecting group can be used as a pro-drugwhereby the carboxy protecting group can be readily cleaved in vivo, forexample by enzymatic hydrolysis, to release the biologically activeparent. Such carboxy protecting groups are well known to those skilledin the art, having been extensively used in the protection of carboxylgroups in the penicillin and cephalosporin fields as described in U.S.Pat. Nos. 3,840,556 and 3,719,667, the disclosures of which are herebyincorporated herein by reference. Representative carboxy protectinggroups are C₁-C₈ loweralkyl (e.g., methyl, ethyl or t-butyl and thelike); arylalkyl such as phenethyl or benzyl and substituted derivativesthereof such as alkoxybenzyl or nitrobenzyl groups and the like;arylalkenyl such as phenylethenyl and the like; aryl and substitutedderivatives thereofsuch as 5-indanyl and the like; dialkylaminoalkylsuch as dimethylaminoethyl and the like); alkanoyloxyalkyl groups suchas acetoxymethyl, butyryloxymethyl, valeryloxymethyl,isobutyryloxymethyl, isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl,1-(pivaloyloxyl)-1-ethyl, 1-methyl-1-(propionyloxy)-1-ethyl,pivaloyloxymethyl, propionyloxymethyl and the like;cycloalkanoyloxyalkyl groups such as cyclopropylcarbonyloxymethyl,cyclobutylcarbonyloxymethyl, cyclopentylcarbonyloxymethyl,cyclohexylcarbonyloxymethyl and the like; aroyloxyalkyl such asbenzoyloxymethyl, benzoyloxyethyl and the like;arylalkylcarbonyloxyalkyl such as benzylcarbonyloxymethyl,2-benzylcarbonyloxyethyl and the like; alkoxycarbonylalkyl orcycloalkyloxycarbonylalkyl such as methoxycarbonylmethyl,cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl and the like;alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl such asmethoxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl,1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl and thelike; aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl,2-(5-indanyloxycarbonyloxy)ethyl and the like;alkoxyalkylcarbonyloxyalkyl such as2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like;arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl andthe like; arylalkenyloxycarbonyloxyalkyl such as2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl and the like;alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and thelike; alkylaminocarbonylaminoalkyl such asmethylaminocarbonylaminomethyl and the like; alkanoylaminoalkyl such asacetylaminomethyl and the like; heterocycliccarbonyloxyalkyl such as4-methylpiperazinylcarbonyloxymethyl and the like;dialkylaminocarbonylalkyl such as dimethylaminocarbonylmethyl,diethylaminocarbonylmethyl and the like;(5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like; and(5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like.

Representative amide carboxy protecting groups are aminocarbonyl andloweralkylaminocarbonyl groups.

Preferred carboxy-protected compounds of the invention are compoundswherein the protected carboxy group is a loweralkyl, cycloalkyl orarylalkyl ester, for example, methyl ester, ethyl ester, propyl ester,isopropyl ester, butyl ester, sec-butyl ester, isobutyl ester, amylester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl esterand the like or an alkanoyloxyalkyl, cycloalkanoyloxyalkyl,aroyloxyalkyl or an arylalkylcarbonyloxyalkyl ester. Preferred amidecarboxy protecting groups are loweralkylaminocarbonyl groups. Forexample, aspartic acid may be protected at the α-C-terminal by an acidlabile group (e.g. t-butyl) and protected at the β-C-terminal by ahydrogenation labile group (e.g. benzyl) then deprotected selectivelyduring synthesis.

B. Modification of ITPs

The manner of producing the modified ITPs of the present invention willvary widely, depending upon the nature of the various elementscomprising the ITP. The synthetic procedures will be selected so as tobe simple, provide for high yields, and allow for a highly purifiedproduct. Normally, the chemically reactive group will be created at thelast stage of the synthesis, for example, with a carboxyl group,esterification to form an active ester. Specific methods for theproduction of modified ITPs of the present invention are describedbelow.

Each ITP selected to undergo the modification with a linker and areactive agent is modified according to the following criteria: if acarboxylic group, not critical for the retention of pharmacologicalactivity is available on the original ITP and no other reactivefunctionality is present on the ITP, then the carboxylic acid is chosenas attachment point for the linker-reactive entity modification. If nocarboxylic acids are available, then other functionalities not criticalfor the retention of pharmacological activity are selected as anattachment point for the linker-reactive entity modification. If severalfunctionalities are available on a an ITP, a combination of protectinggroups will be used in such a way that after addition of thelinker/reactive entity and deprotection of all the protected functionalgroups, retention of pharmacological activity is still obtained. If noreactive functionalities are available on the ITP, synthetic effortswill allow for a modification of the original ITP in such a way thatretention of biological activity and retention of receptor or targetspecificity is obtained.

The chemically reactive entity is placed at a site so that when the ITPis bonded to the blood component, the ITP retains a substantialproportion of the unmodified ITP's activity.

Even more specifically, each ITP selected to undergo the derivatizationwith a linker and a reactive entity will be modified according to thefollowing criteria: if a terminal carboxylic group is available on thetherapeutic peptide and is not critical for the retention ofpharmacological activity, and no other sensitive functional group ispresent on the ITP, then the carboxylic acid will be chosen asattachment point for the linker-reactive entity modification. If theterminal carboxylic group is involved in pharmacological activity, or ifno carboxylic acids are available, then any other sensitive functionalgroup not critical for the retention of pharmacological activity will beselected as the attachment point for the linker-reactive entitymodification. If several sensitive functional groups are available on aITP, a combination of protecting groups will be used in such a way thatafter addition of the linker/reactive entity and deprotection of all theprotected sensitive functional groups, retention of pharmacologicalactivity is still obtained. If no sensitive functional groups areavailable on the therapeutic peptide, synthetic efforts will allow for amodification of the original peptide in such a way that retention ofbiological activity and retention of receptor or target specificity isobtained. In this case the modification will occur at the opposite endof the peptide.

An NHS derivative may be synthesized from a carboxylic acid in absenceof other sensitive functional groups in the therapeutic peptide.Specifically, such a therapeutic peptide is reacted withN-hydroxysuccinimide in anhydrous CH₂Cl₂ and EDC, and the product ispurified by chromatography or recrystallized from the appropriatesolvent system to give the NHS derivative.

Alternatively, an NHS derivative may be synthesized from a ITP thatcontains an amino and/or thiol group and a carboxylic acid. When a freeamino or thiol group is present in the molecule, it is preferable toprotect these sensitive functional groups prior to perform the additionof the NHS derivative. For instance, if the molecule contains a freeamino group, a transformation of the amine into a Fmoc or preferablyinto a tBoc protected amine is necessary prior to perform the chemistrydescribed above. The amine functionality will not be deprotected afterpreparation of the NHS derivative. Therefore this method applies only toa compound whose amine group is not required to be freed to induce apharmacological desired effect. In addition, an NHS derivative may besynthesized from a therapeutic peptide containing an amino or a thiolgroup and no carboxylic acid. When the selected molecule contains nocarboxylic acid, an array of bifunctional linkers can be used to convertthe molecule into a reactive NHS derivative. For instance, ethyleneglycol-bis(succinimydylsuccinate) (EGS) and triethylamine dissolved inDMF and added to the free amino containing molecule (with a ratio of10:1 in favor of EGS) will produce the mono NHS derivative. To producean NHS derivative from a thiol derivatized molecule, one can useN-[-maleimidobutyryloxy]succinimide ester (GMBS) and triethylamine inDMF. The maleimido group will react with the free thiol and the NHSderivative will be purified from the reaction mixture by chromatographyon silica or by HPLC.

An NHS derivative may also be synthesized from a ITP containing multiplesensitive functional groups. Each case will have to be analyzed andsolved in a different manner. However, thanks to the large array ofprotecting groups and bifunctional linkers that are commerciallyavailable, this invention is applicable to any therapeutic peptide withpreferably one chemical step only to derivatize the ITP or two steps byfirst protecting a sensitive group or three steps (protection,activation and deprotection). Under exceptional circumstances only,would one require to use multiple steps (beyond three steps) synthesisto transform a therapeutic peptide into an active NHS or maleimidederivative.

A maleimide derivative may also be synthesized from an ITP containing afree amino group and a free carboxylic acid. To produce a maleimidederivative from a amino derivatized molecule, one can useN-[-maleimidobutyryloxy]succinimide ester (GMBS) and triethylamine inDMF. The succinimide ester group will react with the free amino and themaleimide derivative will be purified from the reaction mixture bycrystallization or by chromatography on silica or by HPLC.

Finally, a maleimide derivative may be synthesized from a therapeuticpeptide containing multiple other sensitive functional groups and nofree carboxylic acids. When the selected molecule contains no carboxylicacid, an array of bifunctional crosslinking reagents can be used toconvert the molecule into a reactive NHS derivative. For instancemaleimidopropionic acid (MPA) can be coupled to the free amine toproduce a maleimide derivative through reaction of the free amine withthe carboxylic group of MPA using HBTU/HOBt/DIEA activation in DMF.

Many other commercially available heterobifunctional crosslinkingreagents can alternatively be used when needed. A large number ofbifunctional compounds are available for linking to entities.Illustrative reagents include: azidobenzoyl hydrazide,N-[4-(p-azidosalicylamino)butyl]-3′-[2′-pyridyldithio)propionamide),bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyltartrate, N-y-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-succinimidyl[4-azidophenyl]-1,3′-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate.

4. Uses of the Modified ITPs

The modified ITPs of the invention find multiple uses including use as atreatment for diabetes, a sedative, a treatment of nervous systemdisorders, use to induce an anxiolytic effect on the CNS, use toactivate the CNS, use for post surgery treatment and as a treatment forinsulin resistance.

A. Diabetes Treatments

The modified ITPs of the invention generally will normalizehyperglycemia through glucose-dependent, insulin-dependent andinsulin-independent mechanisms. As such, the modified ITPs are useful asprimary agents for the treatment of type II diabetes mellitus and asadjunctive agents for the treatment of type I diabetes mellitus.

The use of an effective amount of modified ITPs as a treatment fordiabetes mellitus has the advantage of being more potent than nonmodified ITPs. Since the modified ITPs are move stable in vivo, smalleramounts of the molecule can be administered for effective tratment. Thepresent invention is especially suited for the treatment of patientswith diabetes, both type I and type II, in that the action of thepeptide is dependent on the glucose concentration of the blood, and thusthe risk of hypoglycemic side effects are greatly reduced over the risksin using current methods of treatment.

The present invention also provides for a method for treating diabetesmellitus in an individual, wherein said method comprises providing anamount of modified ITP sufficient to treat diabetes; where thecomposition contains a modified ITP.

B. Treatment Of Nervous System Disorders

The modified ITPs of the invention also find use as a sedative. In oneaspect of the invention, there is provided a method of sedating amammalian subject with an abnormality resulting in increased activationof the central or peripheral nervous system using the modified ITPs ofthe invention. The method comprises administering a modified ITP to thesubject in an amount sufficient to produce a sedative or anxiolyticeffect on the subject. The modified ITP may be administeredintracerebroventriculary, orally, subcutaneously, intramuscularly, orintravenously. Such methods are useful to treat or ameliorate nervoussystem conditions such as anxiety, movement disorder, aggression,psychosis, seizures, panic attacks, hysteria and sleep disorders.

In a related aspect, the invention encompasses a method of increasingthe activity of a mammalian subject, comprising administering a modifiedITP to the subject in an amount sufficient to produce an activatingeffect on the subject. Preferably, the subject has a condition resultingin decreased activation of the central or peripheral nervous system. Themodified ITPs find particular use in the treatment or amelioration ofdepression, schizoaffective disorders, sleep apnea, attention deficitsyndromes with poor concentration, memory loss, forgetfulness, andnarcolepsy, to name just a few conditions in which arousal of thecentral nervous system may be advantageous.

The modified ITPs of the invention may be used to induce arousal for thetreatment or amelioration of depression, schizoaffective disorders,sleep apnea, attention deficit syndromes with poor concentration, memoryloss, forgetfulness, and narcolepsy. The therapeutic efficacy of themodified ITP treatment may be monitored by patient interview to assesstheir condition, by psychological/neurological testing, or byamelioration of the symptoms associated with these conditions. Forexample, treatment of narcolepsy may be assessed by monitoring theoccurrence of narcoleptic attacks. As another example, effects ofmodified ITPs on the ability of a subject to concentrate, or on memorycapacity, may be tested using any of a number of diagnostic tests wellknown to those of skill in art.

C. Post Surgery Treatment

The modified ITPs of the invention may be utilized for post surgerytreatments. A patient is in need of the modified ITPs of the presentinvention for about 1-16 hours before surgery is performed on thepatient, during surgery on the patient, and after the patient's surgeryfor a period of not more than about 5 days.

The modified ITPs of the present invention are administered from aboutsixteen hours to about one hour before surgery begins. The length oftime before surgery when the compounds used in the present inventionshould be administered in order to reduce catabolic effects and insulinresistance is dependent on a number of factors. These factors aregenerally known to the physician of ordinary skill, and include, mostimportantly, whether the patient is fasted or supplied with a glucoseinfusion or beverage, or some other form of sustenance during thepreparatory period before surgery. Other important factors include thepatient's sex, weight and age, the severity of any inability to regulateblood glucose, the underlying causes of any inability to regulate bloodglucose, the expected severity of the trauma caused by the surgery, theroute of administration and bioavailability, the persistence in thebody, the formulation, and the potency of the compound administered. Apreferred time interval within which to begin administration of themodified ITPs used in the present invention is from about one hour toabout ten hours before surgery begins. The most preferred interval tobegin administration is between two hours and eight hours before surgerybegins.

Insulin resistance following a particular type of surgery, electiveabdominal surgery, is most profound on the first post-operative day,lasts at least five days, and may take up to three weeks to normalizeThus, the post-operative patient may be in need of administration of themodified ITPs used in the present invention for a period of timefollowing the trauma of surgery that will depend on factors that thephysician of ordinary skill will comprehend and determine. Among thesefactors are whether the patient is fasted or supplied with a glucoseinfusion or beverage, or some other form of sustenance followingsurgery, and also, without limitation, the patient's sex, weight andage, the severity of any inability to regulate blood glucose, theunderlying causes of any inability to regulate blood glucose, the actualseverity of the trauma caused by the surgery, the route ofadministration and bioavailability, the persistence in the body, theformulation, and the potency of the compound administered. The preferredduration of administration of the compounds used in the presentinvention is not more than five days following surgery.

D. Insulin Resistance Treatment

The modified ITPs of the invention may be utilized to treat insulinresistance independently from their use in post surgery treatment.Insulin resistance may be due to a decrease in binding of insulin tocell-surface receptors, or to alterations in intracellular metabolism.The first type, characterized as a decrease in insulin sensitivity, cantypically be overcome by increased insulin concentration. The secondtype, characterized as a decrease in insulin responsiveness, cannot beovercome by large quantities of insulin. Insulin resistance followingtrauma can be overcome by doses of insulin that are proportional to thedegree of insulin resistance, and thus is apparently caused by adecrease in insulin sensitivity.

The dose of modified ITPs effective to normalize a patient's bloodglucose level will depend on a number of factors, among which areincluded, without limitation, the patient's sex, weight and age, theseverity of inability to regulate blood glucose, the underlying causesof inability to regulate blood glucose, whether glucose, or anothercarbohydrate source, is simultaneously administered, the route ofadministration and bioavailability, the persistence in the body, theformulation, and the potency.

5. Administration of the Modified ITPs

The modified ITPs will be administered in a physiologically acceptablemedium, e.g. deionized water, phosphate buffered saline (PBS), saline,aqueous ethanol or other alcohol, plasma, proteinaceous solutions,mannitol, aqueous glucose, alcohol, vegetable oil, or the like. Otheradditives which may be included include buffers, where the media aregenerally buffered at a pH in the range of about 5 to 10, where thebuffer will generally range in concentration from about 50 to 250 mM,salt, where the concentration of salt will generally range from about 5to 500 mM, physiologically acceptable stabilizers, and the like. Thecompositions may be lyophilized for convenient storage and transport.

The modified ITPs will for the most part be administered orally,parenterally, such as intravascularly (IV), intraarterially (IA),intramuscularly (IM), subcutaneously (SC), or the like. Administrationmay in appropriate situations be by transfusion. In some instances,where reaction of the functional group is relatively slow,administration may be oral, nasal, rectal, transdermal or aerosol, wherethe nature of the conjugate allows for transfer to the vascular system.Usually a single injection will be employed although more than oneinjection may be used, if desired. The modified ITPs may be administeredby any convenient means, including syringe, trocar, catheter, or thelike. The particular manner of administration will vary depending uponthe amount to be administered, whether a single bolus or continuousadministration, or the like. Preferably, the administration will beintravascularly, where the site of introduction is not critical to thisinvention, preferably at a site where there is rapid blood flow, e.g.,intravenously, peripheral or central vein. Other routes may find usewhere the administration is coupled with slow release techniques or aprotective matrix. The intent is that the ITPs be effectivelydistributed in the blood, so as to be able to react with the bloodcomponents. The concentration of the conjugate will vary widely,generally ranging from about 1 pg/ml to 50 mg/ml. The total administeredintravascularly will generally be in the range of about 0.1 mg/ml toabout 10 mg/ml, more usually about 1 mg/ml to about 5 mg/ml.

By bonding to long-lived components of the blood, such asimmunoglobulin, serum albumin, red blood cells and platelets, a numberof advantages ensue. The activity of the modified ITPs compound isextended for days to weeks. Only one administration need be given duringthis period of time. Greater specificity can be achieved, since theactive compound will be primarily bound to large molecules, where it isless likely to be taken up intracellularly to interfere with otherphysiological processes.

The formation of the covalent bond between the blood component may occurin vivo or ex vivo. For ex vivo covalent bond formation, the modifiedITP is added to blood, serum or saline solution containing human serumalbumin or IgG to permit covalent bond formation between the modifiedITP and the blood component. In a preferred format, the ITP is modifiedwith maleimide and it is reacted with human serum albumin in salinesolution. Once the modified ITP has reacted with the blood component, toform a ITP-protein conjugate, the conjugate may be administered to thepatient.

Alternatively, the modified ITP may be administered to the patientdirectly so that the covalent bond forms between the modified ITP andthe blood component in vivo.

6. Monitoring the Presence of Modified ITPs

The blood of the mammalian host may be monitored for the activity of theITPs and/or presence of the modified ITPs. By taking a portion or sampleof the blood of the host at different times, one may determine whetherthe ITP has become bound to the long-lived blood components insufficient amount to be therapeutically active and, thereafter, thelevel of ITP compound in the blood. If desired, one may also determineto which of the blood components the ITP molecule is bound. This isparticularly important when using non-specific ITPs. For specificmaleimide-ITPs, it is much simpler to calculate the half life of serumalbumin and IgG.

The modified GLPs may be monitored using assays of insulinotropicactivity, HPLC-MS or antibodies directed to ITPs.

A. Assays of Insulinotropic Activity

The present invention concerns modified ITPs derivatives which have aninsulinotropic activity that exceeds or equals the insulinotropicactivity of the non-modified ITPs. The insulinotropic property of acompound may be determined by providing that compound to animal cells,or injecting that compound into animals and monitoring the release ofimmunoreactive insulin (IRI) into the media or circulatory system of theanimal, respectively. The presence of IRI is detected through the use ofa radioimmunoassay which can specifically detect insulin.

Although any radioimmunoassay capable of detecting the presence of IRImay be employed, it is preferable to use a modification of the assaymethod of Albano, J. D. M., et al., (Acta Endocrinol. 70:487-509(1972)). In this modification, a phosphate/albumin buffer with a pH of7.4 is employed. The incubation is prepared with the consecutivecondition of 500 μl of phosphate buffer, 50 μl of perfusate sample orrat insulin standard in perfusate, 100 μl of anti-insulin antiserum(Wellcome Laboratories; 1:40,000 dilution), and 100 μl of [¹²⁵I]insulin, giving a total volume of 750 μl in a 10×75-mm disposable glasstube. After incubation for 2-3 days at 4° C., free insulin is separatedfrom antibody-bound insulin by charcoal separation. The assaysensitivity is generally 1-2 μl U/ml. In order to measure the release ofIRI into the cell culture medium of cells grown in tissue culture, onepreferably incorporates radioactive label into proinsulin. Although anyradioactive label capable of labeling a polypeptide can be used, it ispreferable to use ³H leucine in order to obtain labeling of proinsulin.Labeling can be done for any period of time sufficient to permit theformation of a detectably labeled pool of proinsulin molecules; however,it is preferable to incubate cells in the presence of radioactive labelfor a 60-minute time period. Although any cell line capable ofexpressing insulin can be used for determining whether a compound has aninsulinotropic effect, it is preferable to use rat insulinoma cells, andespecially RIN-38 rat insulinoma cells. Such cells can be grown in anysuitable medium; however, it is preferable to use DME medium containing0.1% BSA and 25 mM glucose.

The insulinotropic property of a modified ITP may also be determined bypancreatic infusion. The in situ isolated perfused rat pancreaspreparation is a modification of the method of Penhos, J. C., et al.(Diabetes 18:733-738 (1969)). In accordance with such a method, fastedrats (preferably male Charles River strain albino rats), weighing350-600 g, are anesthetized with an intraperitoneal injection of AmytalSodium (Eli Lilly and Co., 160 ng/kg). Renal, adrenal, gastric, andlower colonic blood vessels are ligated. The entire intestine isresected except for about four cm of duodenum and the descending colonand rectum. Therefore, only a small part of the intestine is perfused,thus minimizing possible interference by enteric substances withinsulinotropic immunoreactivity. The perfusate is preferably a modifiedKrebs-Ringer bicarbonate buffer with 4% dextran T70 and 0.2% bovineserum albumin (fraction V), and is preferably bubbled with 95% O₂ and 5%CO₂. A nonpulsatile flow, four-channel roller-bearing pump (Buchlerpolystatic, Buchler Instruments Division, Nuclear-Chicago Corp.) ispreferably used, and a switch from one perfusate source to another ispreferably accomplished by switching a three-way stopcock. The manner inwhich perfusion is performed, modified, and analyzed preferably followsthe methods of Weir, G. C., et al., (J. Clin. Investigat. 54:1403-1412(1974)), which is hereby incorporated by reference.

B. HPLC-MS

HPLC coupled with mass spectrometry (MS) with can be utilized to assayfor the presence of peptides and modified peptides as is well known tothe skilled artisan. Typically two mobile phases are utilized: 0.1%TFA/water and 0.1% TFA/acetonitrile. Column temperatures can be vairedas well as gradient conditions. Particular details are outlined in theExample section below.

C. Antibodies

Another aspect of this invention relates to methods for determining theconcentration of the ITPs or their conjugates in biological samples(such as blood) using antibodies specific to the ITPs and to the use ofsuch antibodies as a treatment for toxicity potentially associated withsuch ITPs or conjugates. This is advantageous because the increasedstability and life of the ITPs in vivo in the patient might lead tonovel problems during treatment, including increased possibility fortoxicity. The use of anti-ITP antibodies, either monoclonal orpolyclonal, having specificity for particular ITPs, can assist inmediating any such problem. The antibody may be generated or derivedfrom a host immunized with the particular modified ITP, or with animmunogenic fragment of the agent, or a synthesized immunogencorresponding to an antigenic determinant of the agent. Preferredantibodies will have high specificity and affinity for native,derivatized and conjugated forms of the modified ITP. Such antibodiescan also be labeled with enzymes, fluorochromes, or radiolables.

Antibodies specific for modified ITPs may be produced by using purifiedITPs for the induction of derivatized ITP-specific antibodies. Byinduction of antibodies, it is intended not only the stimulation of animmune response by injection into animals, but analogous steps in theproduction of synthetic antibodies or other specific binding moleculessuch as screening of recombinant immunoglobulin libraries. Bothmonoclonal and polyclonal antibodies can be produced by procedures wellknown in the art.

The antibodies may be used to monitor the presence of ITP petides in theblood stream. Blood and/or serum samples may be analyzed by SDS-PAGE andwestern blotting. Such techniques permit the analysis of the blood orserum to determine the bonding of the modified ITPs to blood components.

The anti-therapeutic agent antibodies may also be used to treat toxicityinduced by administration of the modified ITP, and may be used ex vivoor in vivo. Ex vivo methods would include immuno-dialysis treatment fortoxicity employing anti-therapeutic agent antibodies fixed to solidsupports. In vivo methods include administration of anti-therapeuticagent antibodies in amounts effective to induce clearance ofantibody-agent complexes.

The antibodies may be used to remove the modified ITPs and conjugatesthereof, from a patient's blood ex vivo by contacting the blood with theantibodies under sterile conditions. For example, the antibodies can befixed or otherwise immobilized on a column matrix and the patient'sblood can be removed from the patient and passed over the matrix. Themodified ITPs will bind to the antibodies and the blood containing a lowconcentration of the ITP, then may be returned to the patient'scirculatory system. The amount of modified ITP removed can be controlledby adjusting the pressure and flow rate. Preferential removal of themodified ITPs from the plasma component of a patient's blood can beeffected, for example, by the use of a semipermeable membrane, or byotherwise first separating the plasma component from the cellularcomponent by ways known in the art prior to passing the plasma componentover a matrix containing the anti-therapeutic antibodies. Alternativelythe preferential removal of ITP-conjugated blood cells, including redblood cells, can be effected by collecting and concentrating the bloodcells in the patient's blood and contacting those cells with fixedanti-ITP antibodies to the exclusion of the serum component of thepatient's blood.

The anti-ITP antibodies can be administered in vivo, parenterally, to apatient that has received the modified ITP or conjugates for treatment.The antibodies will bind the ITP compounds and conjugates. Once bound,the ITP activity will be hindered if not completely blocked therebyreducing the biologically effective concentration of ITP compound in thepatient's bloodstream and minimizing harmful side effects. In addition,the bound antibody-ITP complex will facilitate clearance of the ITPcompounds and conjugates from the patient's blood stream.

The invention having been fully described is now exemplified by thefollowing non-limiting examples.

EXAMPLES

General

Solid phase peptide syntheses of the insulinotropic peptides on a 100μmole scale was performed using manual solid-phase synthesis and aSymphony Peptide Synthesizer using Fmoc protected Rink Amide MBHA resin,Fmoc protected amino acids,O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) in N,N-dimethylformamide (DMF) solution and activation withN-methyl morpholine (NMM), and piperidine deprotection of Fmoc groups(Step 1). When required, the selective deprotection of the Lys(Aloc)group was performed manually and accomplished by treating the resin witha solution of 3 eq of Pd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc(18:1:0.5) for 2 h (Step 2). The resin was then washed with CHCl₃ (6×5mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). In someinstances, the synthesis was then re-automated for the addition of oneAEEA (aminoethoxyethoxyacetic acid) group, the addition of acetic acidor the addition of a 3-maleimidopropionic acid (MPA) (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The products were purified by preparative reversed phasedHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at 214 and 254nm. Purity was determined 95% by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization.

Example 1 Preparation of Tyr³²-Exendin 4(1-32)-NH₂

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Glu-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Tyr-amide

Solid phase peptide synthesis of the analog on a 100 μmole scale isperformed using manual solid-phase synthesis and a Symphony PeptideSynthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc protectedamino acids, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF) solution andactivation with N-methyl morpholine (NMM), and piperidine deprotectionof Fmoc groups (Step 1). Resin cleavage and product isolation isperformed using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 2). The product is purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired peptidein >95% purity, as determined by RP-HPLC.

Example 2 Preparation of Tyr³¹-Exendin-4(1-31)

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Glu-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Tyr-amide

Solid phase peptide synthesis of the analog on a 100 μmole scale isperformed using manual solid-phase synthesis and a Symphony PeptideSynthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc protectedamino acids, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF) solution andactivation with N-methyl morpholine (NMM), and piperidine deprotectionof Fmoc groups (Step 1). Resin cleavage and product isolation isperformed using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 2). The product is purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired peptidein >95% purity, as determined by RP-HPLC.

Example 3 Preparation of Exendin-4(9-39)-NH₂

Asp-Leu-Ser-Lys-Gin-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asu-Gly-Gly-Pro-Ser-Ser-Gly-Aly-Pro-Pro-Pro-Ser-amide

Solid phase peptide synthesis of the analog on a 100 μmole scale isperformed using manual solid-phase synthesis and a Symphony PeptideSynthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc protectedamino acids, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF) solution andactivation with N-methyl morpholine (NMM), and piperidine deprotectionof Fmoc groups (Step 1). Resin cleavage and product isolation isperformed using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 2). The product is purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired peptidein >95% purity, as determined by RP-HPLC.

Example 4 Preparation of GLP-1 (1-36)-Lys³⁷(ε-MPA)-NH₂.5TFA

His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(ε-MPA)-NH₂.5TFA

The modified GLP-1 peptide is synthesized by linking off the amino groupof the added Lysine residue as shown in the schematic diagram below.

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH,Boc-His(N-Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Boc-His(N-Trt)-OH (step 1)

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.

Example 5 Preparation of GLP-1 (1-36)-Lys³⁷(ε-AEEA-AEEA-MPA)-NH₂.5TFA

His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(ε-AEEA-AEEA-MPA)-NH₂.5TFA

The modified GLP-1 peptide is synthesized by linking off the amino groupof the added Lysine residue as shown in the schematic diagram below.

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH,Boc-His(N-Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Boc-His(N-Trt)-OH (step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the two AEEA (aminoethoxyethoxyacetic acid) groupsand the 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reverse phase HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization, ESI-MS m/z forC₁₇₄H₂₆₅N₄₄O₅₆ (MH⁺), calcd 3868, found [M+H₂]²⁺ 1934, [M+H₃]³⁺ 1290,[M+H₄]⁴⁺ 967.

Example 6 Preparation of GLP-1 (7-36)-Lys³⁷(ε-MPA)-NH₂.4TFA

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(ε-MPA)-NH₂.4TFA

The modified GLP-1 peptide is synthesized by linking off the ε-Nterminus of the added Lysine residue as described below.

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Alock)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH,Boc-His(N-Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.

Example 7 Preparation of GLP-1 (7-36)-Lys³⁷(ε-AEEA-AEEA-MPA)-NH₂.4TFA

His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(ε-AEEA-AEEA-MPA)-NH₂.4TFA

The modified GLP-1 peptide is synthesized by linking off the ε-Nterminus of the added Lysine residue as described below.

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH,Boc-His(N-Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the two AEEA (aminoethoxyethoxyacetic acid) groupsand the 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reverse phase HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization.

Example 8 Preparation of D-Ala⁸ GLP-1 (7-36)-Lys³⁷(ε-MPA)-NH₂.4TFA

His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(ε-MPA)-NHH₂.4TFA

D-Ala⁸ GLP-1 (7-36) amide was synthesized as shown in the schematicdiagram below.

A. Preparation of D-Ala⁸-GLP-1 (7-36) Amide

Solid phase peptide synthesis of the GLP-1 analog on a 100 μmole scaleis performed using manual solid-phase synthesis and a Symphony PeptideSynthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc protectedamino acids, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF) solution andactivation with N-methyl morpholine (NMM), and piperidine deprotectionof Fmoc groups (Step 1). Resin cleavage and product isolation isperformed using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 2). The product is purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired peptidein >95% purity, as determined by RP-HPLC.

The modified GLP-1 peptide is synthesized by linking off the ε-Nterminus of the added Lysine residue as shown in the schematic diagrambelow.

B. Preparation of D-Ala⁸-GLP-1 (7-36)-Lys³⁷ (E-MPA) Amide

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Ring Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-d-Ala-OH,Boc-His(N-Trt)-OH (Step1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.

Example 9 Preparation of D-Ala⁸ GLP-1(7-36)-Lys³⁷(ε-AEEA-AEEA-MPA)-NH₂.4TFA

His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(ε-AEEA-AEEA-MPA)-NH₂.4TFA

The modified GLP-1 peptide is synthesized by linking off the □-Nterminus of the added Lysine residue as shown in the schematic diagrambelow.

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-d-Ala-OH,Boc-His(N-Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the two AEEA (aminoethoxyethoxyacetic acid) groupsand the 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reverse phase HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization.

Example 10 Preparation of Exendin-4 (1-39)-Lys⁴⁰(ε-MPA)-NH₂

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(ε-MPA)-NH₂.5TFA

Exendin4 is synthesized as shown in the schematic below.

A. Preparation of Exendin 4

Solid phase peptide synthesis of Exendin-4 on a 100 μmole scale isperformed using manual solid-phase synthesis and a Symphony PeptideSynthesizer using Fmoc protected Rink Amide MBHA resin The followingprotected amino acids are sequentially added to the resin:Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH,Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH,Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH,Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH,Boc-His(Trt)-OH. They are dissolved in N,N-dimethylformamide (DMF) and,according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). Resin cleavage andproduct isolation is performed using 85% TFA/5% TIS/5% thioanisole and5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 2). Theproduct is purified by preparative reversed phased HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at 214 and 254 nm to afford thedesired peptide in >95% purity, as determined by RP-HPLC.

B. Preparation of Modified Exendin 4 (SEQ ID NO:18)

The modified exendin-4 peptide is synthesized by linking off the ε-Nterminus of the added Lysine residue as shown in the schematic diagrambelow.

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH,Fmoc-Trp-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH,Fmoc-Arg(Bpf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Boc-His(Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.

Example 11 Preparation of Modified Exendin-4(1-39)-Lys⁴⁰(E-AEEA-AEEA-MPA)-NH₂.5TFA

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(ε-AEEA-AEEA-MPA)-NH₂.5TFA

The modified exendin-4 peptide is synthesized by linking off the ε-Nterminus of the added Lysine residue as shown in the schematic diagrambelow.

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH,Fmoc-Trp-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH,Fmoc-Arg(Bpf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Boc-His(Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the two AEEA (aminoethoxyethoxyacetic acid) groupsand the 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reverse phase HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization.

Example 12 Preparation of Exendin-3 (1-39)-Lys⁴⁰(ε-MPA)-NH₂.5TFA

His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(ε-MPA)-NH₂.5TFA

A Preparation of Exendin 3

The exendin-3 peptide first is synthesized as described in the schematicbelow.

Solid phase peptide synthesis of Exendin 3 on a 100 μmole scale isperformed using manual solid-phase synthesis and a Symphony PeptideSynthesizer using Fmoc protected Rink Amide MBHA resin The followingprotected amino acids are sequentially added to the resin:Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH,Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH,Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH,Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH,Boc-His(Trt)-OH. They are dissolved in N,N-dimethylformamide (DMF) and,according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (VN) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). Resin cleavage andproduct isolation is performed using 85% TFA/5% TIS/5% thioanisole and5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 2). Theproduct is purified by preparative reversed phased HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at 214 and 254 nm to afford thedesired peptide in >95% purity, as determined by RP-HPLC.

B. Preparation of Modified Exendin 3

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH,Fmoc-Trp-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH,Fmoc-Arg(Bpf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Boc-His(Trt)-OH (Step1). The modified exendin 3 is synthesized by linking off the ε-Nterminus of the added lysine residue.

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.

Example 13 Preparation of Exendin-3(1-39)-Lys⁴⁰(ε-AEEA-AEEA-MPA)-NH₂.5TFA

His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(ε-AEEA-AEEA-MPA)-NH₂.5TFA

The modified exendin-3 peptide is synthesized by linking off the ε-Nterminus of the added Lysine residue as described below.

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH,Fmoc-Trp-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH,Fmoc-Arg(Bpf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Boc-His(Trt)-OH (Step1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL. The synthesis was then re-automatedfor the addition of the two AEEA (aminoethoxyethoxyacetic acid) groupsand the 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reverse phase HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization.

Example 14 Preparation of Lys²⁶(ε-MPA)GLP-1(7-36)-NH₂

Solid phase peptide synthesis of the DAC:GLP-1 analog on a 100 μmolescale is performed manually and on a Symphony Peptide Synthesizer usingFmoc protected Rink amide MBHA resin. The following protected aminoacids are sequentially added to the resin: Fmoc-Arg(Pbf)-OH,Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH,Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-lIe-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH,Boc-His(Trt)-OH. They are dissolved in N,N-dimethylformamide (DMF) and,according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). Selectivedeprotection of the Lys(Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Resin cleavage andproduct isolation is performed using 86% TFA/5% TIS/5% H₂O/2%thioanisole and 2% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product is purified by preparative reversed phaseHPLC using a Varian (Rainin) preparative binary HPLC system using aDynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a Dynamax C₁₈,60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ214 and 254 nm to afford the desired DAC in >95%purity, as determined by RP-HPLC.

Example 15 Preparation of GLP-1 (7-36)-EDA-MPA

Solid phase peptide syntheses of the modifiedGLP-1 analog on a 100 μmolescale is performed manually and on a Symphony Peptide Synthesizer SASRIN(super acid sensitive resin). The following protected amino acids aresequentially added to the resin: Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH,Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH,Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gly-OH, Fmco-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His(Trt)-OH. They aredissolved in N,N-dimethylformamide (DMF) and, according to the sequence,activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (Step 1). Thefully protected peptide is cleaved from the resin by treatment with 1%TFA/DCM (Step 2). Ethylenediamine and 3-maleimidopropionic acid are thensequentially added to the free C-terminus (Step 3). The protectinggroups are then cleaved and the product isolated using 86% TFA/5% TIS/5%H₂O/2% thioanisole and 2% phenol, followed by precipitation by dry-icecold Et₂O (Step 4). The product is purified by preparative reversedphase HPLC using a Varian (Rainin) preparative binary HPLC system usinga Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a DynamaxC₁₈, 60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ214 and 254 nm to afford the desired DAC in >95%purity, as determined by RP-HPLC.

Example 16 Preparation of Exendin-4 (1-39)-EDA-MPA

The schematic below illustrates the synthesis of Exendin-4(1-39)-EDA-MPA.

Solid phase peptide syntheses of the modified Exendin4 analog on a 100μmole scale is performed manually and on a Symphony Peptide SynthesizerSASRIN (super acid sensitive resin). The following protected amino acidsare sequentially added to the resin: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH,Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH,Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Boc-His(Trt)-OH. They aredissolved in N,N-dimethylformamide (DMF) and, according to the sequence,activated using 0-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (Step 1). Thefully protected peptide is cleaved from the resin by treatment with 1%TFA/DCM (Step 2). Ethylenediamine and 3-maleimidopropionic acid are thensequentially

35 1 37 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 1 His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe ThrSer Asp Val 1 5 10 15 Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu PheIle Ala Trp Leu 20 25 30 Val Lys Gly Arg Gly 35 2 31 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide 2 His AlaGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 GlnAla Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 3 22PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 3 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp LeuVal 1 5 10 15 Xaa Gly Arg Xaa Gly Arg 20 4 4 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 4 Ser Asp Val Ser 15 5 PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 5 Thr Ser Asp Val Ser 1 5 6 6 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 6 Phe Thr Ser AspVal Ser 1 5 7 7 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 7 Thr Phe Thr Ser Asp Val Ser 1 5 8 8 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide8 Gly Thr Phe Thr Ser Asp Val Ser 1 5 9 9 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 9 Glu Gly Thr PheThr Ser Asp Val Ser 1 5 10 10 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 10 Ala Glu Gly Thr Phe Thr Ser AspVal Ser 1 5 10 11 39 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 11 His Ser Asp Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 1239 PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 12 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met GluGlu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly GlyPro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35 13 31 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide 13 His GlyGlu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Tyr 20 25 30 14 31PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 14 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met GluGlu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly GlyTyr 20 25 30 15 31 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 15 Asp Leu Ser Lys Gln Met Glu Glu Glu AlaVal Arg Leu Met Ile Glu 1 5 10 15 Trp Leu Lys Asn Gly Gly Pro Ser SerGly Ala Pro Pro Pro Ser 20 25 30 16 37 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 16 His Asp Glu PheGlu Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val 1 5 10 15 Ser Ser TyrLeu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu 20 25 30 Val Lys GlyArg Lys 35 17 31 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 17 His Ala Glu Gly Thr Phe Thr Ser Asp ValSer Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala TrpLeu Val Lys Gly Arg Lys 20 25 30 18 40 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 18 His Gly Glu GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala ValArg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly AlaPro Pro Pro Ser Lys 35 40 19 40 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 19 His Ser Asp Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe IleGlu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro SerLys 35 40 20 31 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 20 His Gly Glu Gly Thr Phe Thr Ser Asp LeuSer Lys Glu Met Glu Glu 1 5 10 15 Glu Val Arg Leu Phe Ile Glu Trp LeuLys Asn Gly Gly Pro Tyr 20 25 30 21 30 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 21 His Gly Glu GlyThr Phe Thr Ser Asp Leu Ser Lys Glu Met Glu Glu 1 5 10 15 Glu Val ArgLeu Phe Ile Glu Trp Leu Lys Asn Gly Gly Tyr 20 25 30 22 29 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide22 Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1 510 15 Trp Leu Lys Gly Gly Pro Ser Ser Gly Pro Pro Pro Ser 20 25 23 31PRT Artificial Sequence Description of Artificial Sequence SyntheticPeptide 23 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met GluGlu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly GlyXaa 20 25 30 24 31 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 24 Asp Leu Ser Lys Gln Met Glu Glu Glu AlaVal Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn Gly Gly Pro Ser SerGly Ala Pro Pro Pro Xaa 20 25 30 25 37 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 25 His Asp Glu PheGlu Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val 1 5 10 15 Ser Ser TyrLeu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu 20 25 30 Val Lys GlyArg Xaa 35 26 37 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 26 His Asp Glu Phe Glu Arg His Ala Glu GlyThr Phe Thr Ser Asp Val 1 5 10 15 Ser Ser Tyr Leu Glu Gly Gln Ala AlaLys Glu Phe Ile Ala Trp Leu 20 25 30 Val Lys Gly Arg Xaa 35 27 31 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide27 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 510 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 2530 28 31 PRT Artificial Sequence Description of Artificial SequenceSynthetic Peptide 28 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser TyrLeu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val LysGly Arg Xaa 20 25 30 29 31 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 29 His Xaa Glu Gly Thr Phe Thr SerAsp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe IleAla Trp Leu Val Lys Gly Arg Xaa 20 25 30 30 31 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 30 His Xaa Glu GlyThr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala AlaLys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25 30 31 40 PRTArtificial Sequence Description of Artificial Sequence Synthetic Peptide31 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 2025 30 Ser Gly Ala Pro Pro Pro Ser Xaa 35 40 32 40 PRT ArtificialSequence Description of Artificial Sequence Synthetic Peptide 32 His GlyGlu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 GluAla Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 SerGly Ala Pro Pro Pro Ser Xaa 35 40 33 40 PRT Artificial SequenceDescription of Artificial Sequence Synthetic Peptide 33 His Ser Asp GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala ValArg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly AlaPro Pro Pro Ser Xaa 35 40 34 40 PRT Artificial Sequence Description ofArtificial Sequence Synthetic Peptide 34 His Ser Asp Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe IleGlu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro SerXaa 35 40 35 32 PRT Artificial Sequence Description of ArtificialSequence Synthetic Peptide 35 His Gly Glu Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Gly Pro Xaa 20 25 30

We claim:
 1. A compound of D-Ala⁸GLP-1 (7-36)-Lys³⁷(ε-MPA)-NH₂.
 2. Acomposition comprising a compound as claimed in claim 1 in associationwith a pharmaceutically acceptable carrier.